Anal. Chem. 1997, 69, 1326-1331
Determination of Glucose in Submicroliter Samples by CE-LIF Using Precolumn or On-Column Enzymatic Reactions Zhe Jin, Rong Chen, and Luis A. Colo´n*
Department of Chemistry, Natural Science and Mathematics Complex, State University of New York at Buffalo, Buffalo, New York 14260-3000
Two enzymatic reactions combined with capillary electrophoresis (CE) are used to determine glucose contained in sample volumes of e500 nL. In the first enzymatic reaction, glucose is oxidized in the presence of glucose oxidase producing hydrogen peroxide, which reacts quantitatively with the fluorogenic compound homovanillic acid catalyzed by the enzyme peroxidase. The second reaction generates a fluorescent species that is proportional to the glucose concentration. The reaction product is determined by CE using laser-induced fluorescence (LIF) as the detection mode. The overall reaction scheme is faster than commonly used precolumn derivatization procedures and can be performed using very small sample quantities. Alternatively, the enzymatic reactions can be performed on-column, similar to the electrophoretically mediated microanalysis approach, accommodating sample quantities in the nanoliter range. The on-column reaction is a simple and practical approach for the determination of glucose contained in low-volume samples by CE-LIF, where samples are injected directly into the capillary column without any pretreatment. However, sample handling and detectability of the precolumn approach proved to be superior. Determination of glucose using the precolumn and on-column reactions showed detection limits of 50 and 800 nM, respectively. The methods were shown to be linear in the range tested, 1-100 µM and 100 nM-30 µM, for the on-column and precolumn reactions, respectively. The reproducibility for each scheme was 9). To investigate how the fluorescence intensity is affected by the pH of the electrolyte used for detection, we performed the (17) (a) Guilbault, G. G.; Brignac, P. J., Jr.; Juneau, M. Anal. Chem. 1968, 40, 1256-1263. (b) Zaitsu, K.; Ohkura, Y. Anal. Biochem. 1980, 109, 109113. (c) Cathcart, R.; Schwiers, E.; Ames, B. N. Anal. Biochem. 1983, 134, 11-116. (18) Genfa, Z.; Dasgupta, P. Anal. Chem. 1992, 64, 517-522. (19) Kiang, S. W.; Kuan, J. W.; Kuan, S. S.; Guilbault, G. G. Clin. Chem. 1976, 22, 1378-1382.
1328 Analytical Chemistry, Vol. 69, No. 7, April 1, 1997
Figure 1. Dependence of the signal intensity on the pH of the CE running electrolyte.
enzymatic reactions using a phosphate buffer system with a pH of 7 (a final concentration of 5 mM) and a 3 µM glucose solution. The pH of the detection electrolyte was adjusted and the fluorescence intensity monitored by CE-LIF. Figure 1 illustrates how the relative fluorescence intensity depends on the pH of the electrolyte used for detection. The relative fluorescence intensity reaches its maximum at a pH close to 9.3. We also performed the enzymatic reactions at different pHs (6-9) and monitored the reaction product at pH 9.3. Maximum signal was observed when the reactions were performed at pH 7. Thus, we performed the enzymatic reactions at a more favorable pH condition (pH 7) and then analyzed the reaction products by CE-LIF using a detection electrolyte with a pH of 9.3. Although in principle either the end point assay or the rate (kinetic) assay method can be used to determine glucose, we selected the end point approach because of its higher precision and lower susceptibility to small changes in temperature, enzyme activity, time of measurements, and incubation.20 Figure 2 depicts the dependence of the fluorescence intensity on the reaction time at four different temperatures. As the incubation temperature is increased, less time is required to obtain the maximum signal intensity. For example, when the reaction mixture is incubated at 40 °C, the maximum signal is obtained in less than 20 min. When the reaction is performed at room temperature, the maximum signal intensity is achieved ∼35 min after initiating the reaction. All subsequent experiments involving the enzymatic reactions prior to injection were performed in a phosphate buffer (5 mM final concentration) at pH ∼7 at room temperature. The reaction products were analyzed by CE-LIF using 10 mM of the running electrolyte (pH ∼9.3). Two schemes are possible for the detection of glucose using the above enzymatic reactions. In the first one, the enzymatic (20) Harris, D. A.; Bashford, C. L. Spectrophotometry & Spectrofluorimetry: a practical approach; IRL Press: Washington, DC, 1987, pp 54-55.
Figure 2. Effect of reaction time and temperature on the signal response.
Figure 3. Electropherograms of the precolumn reactions corresponding to a blank (A) and 1 µM glucose (B). CE conditions were 10 s pressure injection (0.5 psi), separation voltage of 20 kV, 10 mM borate (pH 9.2) running buffer, fused-silica capillary (50 µm i.d., 27 cm length, injector to detector 20 cm), and LIF detection (325 nm exitation and emission collected through 380 nm filter).
reactions are performed prior to injection into the CE system (precolumn reactions). This procedure provides the opportunity to perform the enzymatic reactions and detection at optimized pH conditions. In the second scheme, the reactions are performed on-column, which allows the use of very small sample quantities without the need for any precolumn procedure. In this approach, however, the reaction and the detection are performed under the same pH conditions, which compromises enzyme activity and maximum fluorescence intensity of HVAox. Nevertheless, both schemes were explored. CE-LIF Precolumn Reactions. A typical electropherogram corresponding to a precolumn reaction of 1 µM glucose at room temperature is shown in Figure 3. A control run (blank) gave a small fluorescence signal, which was easily subtracted from the sample signal. This blank signal arises from mixing HVA and peroxidase and can be attributed to impurities in the commercial
peroxidase reagent which oxidizes HVA.17a The product of the reaction is analyzed by CE-LIF. The reproducibility in the migration time for HVAox was ∼0.2% RSD (n ) 18). The peak height reproducibility associated with the reaction of 1 µM glucose for five different reactions was
